Method for controlling culture of lactic bacteria

ABSTRACT

A novel method for controlling culture of lactic bacteria, which enables the state of bacteria to be controlled in a simple and rapid manner in the step of cultivation in the production of various products utilizing lactic bacteria. The method is characterized in that, in the step of cultivating lactic bacteria, the intensity of the infrared absorption assignable to dissociable lactic acid and that of the infrared absorption assignable to nondissociable lactic acid in the culture are measured by FT-IR spectrometry and the pH value of the culture and/or the lactic acid concentration of the culture are calculated based on the measurements. The method according to another embodiment is characterized in that, in the above method for controlling culture of lactic bacteria, the intensity of the infrared absorption assignable to glucose as a substrate or an alcoholic C-O group of lactose is measured and the glucose concentration or lactose concentration is calculated based on these measurements. The method according to a further embodiment is characterized in that the intensity of the infrared absorption is measured in-line by the ATR-FT-IR spectrometry and, based on the measurements, the state of culture of lactic bacteria is automatically monitored and controlled.

TECHNICAL FIELD

The present invention relates to a method for controlling culture oflactic bacteria in the step of cultivation in the production of variousproducts utilizing lactic bacteria; more specifically, the presentinvention relates to a method for controlling the step of culture oflactic bacteria particularly utilizing the ATR-FT-IR spectrometry,particularly a novel method for controlling culture of lactic bacteria,which enables the state of culture of lactic bacteria to be monitoredand controlled in a simple and rapid manner, by employing theconcentration of glucose or lactose as a substrate, the concentration oflactic acid as a culture product of lactic bacteria, and the acidity andthe pH value of a fermented liquid as indexes.

TECHNICAL BACKGROUND

Generally, in the production of various products utilizing lacticbacteria such as fermented milk, a method for controlling a fermentationof the culture, namely, a method for investigating the state of thefermentation proceeded and determining the completion of thefermentation, is carried out by employing the concentration of asaccharide as a substrate, the concentration of lactic acid as a productformed following fermentation, and values of the acidity and the pHvalue of a fermented liquid, as indexes in the control of the step ofculture. In order to obtain these values, conventionally a fermentedliquid has been sampled once at a proper step, and then have beenanalyzed quantitatively regarding each item according to an ordinaryprocedure, utilizing, for example, a neutralization titration method.According to such a method, however, there occurs a problem of a largeaccidental error in titration for measuring each index value; inaddition, since much time and many hands are required, it has not beenpossible to control the step of cultivation in a simple and rapidmanner.

Moreover, recently there have been found examples investigating apossibility of a method of more reliable and simple on-line measurement;however, they are only for obtaining basic knowledge about on-linemeasurement in the step of the fermentation of specific products and arestill matters of basic investigation; and no example actuallyinvestigating whether it is possible or not to control the step ofcultivation of lactic bacteria according to such methods of measurementhas been reported, and hence it has been desired keenly in the fieldconcerned to develop a new method for controlling the step ofcultivation of lactic bacteria utilizing a method of more reliable andsimple measurement.

As described above, it has been desired keenly to develop a method formeasuring the concentration of a saccharide, the concentration of lacticacid, acidity and a pH value as indexes for controlling the state ofculture of lactic bacteria in a rapid, simple and precise manner,particularly, to develop a method of in-line measurement and a methodfor controlling culture of lactic bacteria based thereon.

The present inventors have engaged in assiduous studies with a view todeveloping such a new method for controlling culture of lactic bacteria,and as a result have found that the infrared absorption spectrum of afermented liquid is measured by the FT-IR spectrometry (Fouriertransform infrared spectroscopy) and that of the infrared absorptionassignable to dissociable lactic acid, the intensity of the infraredabsorption assignable to nondissociable lactic acid in the culture, theintensity of the infrared absorption assignable to an alcoholic C-Ogroup of glucose and the intensity of the infrared absorption assignableto an alcoholic C-O group of lactose are measured, and the lactic acidconcentration, the pH value, the glucose concentration and the lactoseconcentration of the culture can be measured based on these measurementsand that the state of culture of lactic bacteria can be monitored andcontrolled in a simple and rapid manner, by employing these measurementsas indexes, which has led to the completion of the present invention. Inaddition, they have found that, of FT-IR methods, the ATR-FT-IRspectrometry, a method for measuring a whole reflective spectrum,enables the numerical values of various indexes to be measured and,based on the measurements, the culture of lactic bacteria isautomatically controlled.

SUMMARY OF THE INVENTION

It is the objective of the present invention to provide a novel methodfor controlling culture of lactic bacteria, which enables the state ofculture of lactic bacteria to be controlled in a simple and rapid mannerin the step of cultivation in the production of various productsutilizing lactic bacteria.

The present invention relates to a method for controlling culture oflactic bacteria, characterized in that the method comprises measuring,in the step of cultivating lactic bacteria, the intensity of theinfrared absorption assignable to dissociable lactic acid and that ofthe infrared absorption assignable to nondissociable lactic acid in theculture by the FT-IR spectrometry, and calculating the pH value of theculture and/or the lactic acid concentration of the culture according tothese measurements. The method according to another embodiment is amethod for controlling culture of lactic bacteria, characterized inthat, in the above method for controlling culture of lactic bacteria,the intensity of the infrared absorption assignable to an alcoholic C-Ogroup of glucose or lactose as a substrate is measured and the glucoseconcentration or the lactose concentration is calculated based on thesemeasurements; and the method according to a further embodiment is amethod for controlling culture of lactic bacteria, characterized in thatthe intensity of the infrared absorption is measured in-line by theATR-FT-IR spectrometry and, based on the measurements, the state ofculture of lactic bacteria is automatically monitored and controlled.

According to the present invention, it becomes possible, in theproduction of products utilizing lactic bacteria such as fermented milk,to measure various index values needed for controlling the step ofcultivation in a rapid, simple and precise manner, and, based on themeasurements, the state of culture of lactic bacteria in the step ofcultivation can be monitored and controlled in a simple and rapidmanner. Besides, the above various index values can be measured in-lineby the ATR-FT-IR spectrometry and the step of culture of lactic bacteriacan be controlled automatically.

DISCLOSURE OF THE INVENTION

It is an objective of the present invention to provide a method forcontrolling the step of culture in the production of various productsutilizing lactic bacteria such as fermented milk.

It is another objective of the present invention to provide a novelmethod for controlling culture of lactic bacteria, which enables variousindex values controlling culture to be measured in a rapid and simplemanner in the step of culture of lactic bacteria and the state ofculture of lactic bacteria to be monitored and controlled in a simpleand rapid manner.

It is still another objective of the present invention to provide amethod for controlling culture of lactic bacteria automatically in asimple and rapid manner by measuring the above index values particularlyin-line in the above step of cultivating lactic bacteria;

The present invention dissolving the above problems is a method forcontrolling culture of lactic bacteria, characterized in that the methodcomprises measuring, in the step of cultivating lactic bacteria, theintensity of the infrared absorption assignable to dissociable lacticacid and that of the infrared absorption assignable to nondissociablelactic acid in the culture by the FT-IR spectrometry, and calculatingthe pH value of the culture and/or the lactic acid concentration of theculture according to these measurements.

In addition, the method according to another embodiment is a method forcontrolling culture of lactic bacteria, characterized in that, in theabove method for controlling culture of lactic bacteria, the intensityof the infrared absorption assignable to an alcoholic C-O group ofglucose or lactose as a substrate is measured and the glucoseconcentration or the lactose concentration is calculated based on thesemeasurements. The method according to a further embodiment is a methodfor controlling culture of lactic bacteria, characterized in that theintensity of the infrared absorption is measured in-line by theATR-FT-IR spectrometry. The method according to a further embodiment isa method for controlling culture of lactic bacteria, characterized inthat the intensity of the infrared absorption assignable to dissociablelactic acid is the intensity of the infrared absorption at about 1575cm⁻¹ and the intensity of the infrared absorption assignable tonondissociable lactic acid is the intensity of the infrared absorptionat about 1725 cm⁻¹ . Further, the method according to a furtherembodiment is a method for controlling culture of lactic bacteria,characterized in that the intensity of the infrared absorptionassignable to an alcoholic C-O group of glucose is the intensity of theinfrared absorption at about 1080 cm⁻¹ or about 1035 cm⁻¹ and theintensity of the infrared absorption assignable to an alcoholic C-Ogroup of lactose is the intensity of the infrared absorption at about1075 cm⁻¹ or about 1042 cm⁻¹.

The wave-numbers specified here include a range of about±10 cm⁻¹.

Next, the present invention will be described in more detail.

Lactic bacteria according to the present invention are dairy lacticbacteria generally utilized for the dairy industry, and, for example,lactic bacteria such as the Lactobacillus genus, the streptococcusgenus, the Lactococcus genus and the Leuconostoc genus can beexemplified as typical ones; in addition, bacteria capable ofcontrolling cultivation similarly with the saccharide concentration, thelactic acid concentration and a pH value capable of being measuredaccording to the method of the present invention as indexes are includednaturally in lactic bacteria according to the present invention as onescapable of being utilized in the same manner, and examples thereofinclude the Bifidobacterium genus, the Pediococcus genus and theSporolactobacillus genus.

The step of culture of lactic bacteria according to the presentinvention typically means the step of fermentation of various productsutilizing lactic bacteria such as fermented milk; however, steps offermentation and cultivation including lactic bacteria such as the stepof cultivation aiming at culture of lactic bacteria themselves aretargeted irrespective of kind.

The FT-IR (Fourier transform infrared spectroscopy) spectrometry,particularly the ATR (attenuated total reflection)-FT-IR spectrometrydescribed herein comprises introducing light of the infrared region froma sample into a light interference meter, measuring the intensity oflight coming therefrom as a function of the moving distance of a movablemirror and obtaining a spectrum by the Fourier transform; in particular,the ATR-FT-IR spectrometry is a method known as spectrometry measuringthe change of the intensity of the whole reflected light when the angleof incidence of light with a constant wavelength is changed.

The in-line method according to the present invention means a method formeasuring data by fixing a probe (sensor part) of an infrared absorptionspectrum measuring device according to the present invention directly toa culture tank for performing culture of lactic bacteria, which enablesthe data to be measured directly by the culture tank without employing astep of performing sampling once during the cultivation of lacticbacteria as in a conventional method. Moreover, it is substantiallydiscriminated from an indirect method guessing the lactic acidconcentration from the measurement of the amount of consumption of asubstrate such as lactose and glucose as in a conventional method.

In the present invention, the infrared absorption spectrum of a culturebroth of lactic bacteria aiming at controlling a fermentation of theculture was measured by the FT-IR spectrometry, and the correlationbetween the intensity of a specific wave-number and values such as theglucose concentration, the lactose concentration, the lactic acidconcentration, acidity and a pH value was examined specifically.

It has been revealed as a result of it that there exists a strongcorrelation (coefficient of correlation: 0.999) between the glucoseconcentration and the absorption intensity assignable to an alcoholicC-o group of glucose at 1080 cm⁻¹ or 1035 cm⁻¹ according to a testemploying an aqueous glucose solution as a standard liquid (see FIG. 1and FIG. 2). Besides, it has been revealed that, also in the case ofperforming measurement in-line by fixing a probe of an infraredabsorption spectrum measuring device directly to a fermentation tank forculturing lactic bacteria, there exist strong correlations at respectivewave-numbers (coefficient of correlation: 0.992 at 1080 cm⁻¹, 0.998 at1035 cm⁻¹), and that the glucose concentration can be measured from theintensity of the IR absorption (see FIG. 5).

Next, it has been revealed that there exists a strong correlation(coefficient of correlation: 0.998) between the lactose concentrationand the absorption intensity assignable to an alcoholic C-O group oflactose at 1075 cm⁻¹ or 1042 cm⁻¹ according to a test employing anaqueous lactose solution as a standard liquid (see FIG. 3). Besides, ithas been revealed that, also in the case of performing measurementin-line by fixing a probe of an infrared absorption spectrum measuringdevice directly to a fermentation tank for culturing lactic bacteria,there exist strong correlations at respective wave-numbers (coefficientof correlation: 0.995 at 1075 cm⁻¹, 0.998 at 1042 cm⁻¹), and that thelactose concentration can be measured from the intensity of the IRabsorption (see FIG. 6).

Next, it has been revealed that there exists an excellent correlationbetween the lactose concentration and the absorption intensityassignable to a C=O group of carboxylic acid at 1725 cm⁻¹, theabsorption intensity assignable to a C-O group of carboxylic acid at1237 cm⁻¹ and the absorption intensity assignable to an alcoholic C-Ogroup at 1132 cm⁻¹ according to a test employing an aqueous lactic acidsolution as a standard liquid (see FIG. 4); however, in the case ofperforming measurement in-line by fixing a probe directly to afermentation tank, there is observed no linear relation between theabsorption intensity of any wave-number and the lactic acidconcentration. Hence, it has been found as a result of investigatingthis point variously that the lactic acid concentration can becalculated by taking note of the dissociation ratio of lactic acid andemploying the double regression method combining the intensity of theinfrared absorption assignable to dissociable lactic acid and theintensity of the infrared absorption assignable to nondissociable lacticacid. Moreover, it has been found as a simpler method of calculationthat the lactic acid concentration can be calculated by performing anamendment on a ratio of depth of creeping into a measuring sample, afunction of a wave-number of infrared rays, at each intensity in the ATRspectrometry.

Moreover, it has been found that acidity and a pH value can becalculated precisely from the dissociation ratio of lactic acid byapplying the Henderson-Hasselbalch equation.

That is, an explanation will be made employing Example 1 to be describedlater as an example; in the case that the pH when the dissociation ratioof an electrolyte is 50% is made pKa, the dissociation ratio (%) is asbelow by varying the Henderson-Hasselbalch equation, pH=pKa+log[dissociation concentration]/[nondissociation concentration]:

dissaciation ratio={10.sup.(pH-pKa) /(10.sup.(pH-pKa) +1)}×100; andsince pKa of lactic acid is 3.86, the relation between the pH and thedissociation ratio is as below:

dissociaton ratio={10.sup.(pH-3.86) (10.sup.(pK-3.86) +1)}×100. Sincethe pH in the step of cultivation in the logosa medium in Example 1 tobe described later is varied to from 6.8 to 3.5, the dissociation ratiois varied to from 99 to 33%.

With a view to confirming the absorption wave-numbers of dissociablelactic acid and nondissociable lactic acid, the infrared absorptionspectrum of an 1% lactic acid standard liquid varying the pH thereoffrom 2 to 7 is shown in FIG. 7.

An equation for obtaining the lactic acid concentration (L)(g/l) was ledby selecting a wave-number at 1575 cm⁻¹ as an absorption wave-numberwith a small noise due to impurities and a large relative change fordissociable lactic acid and a wave-number at 1725 cm⁻¹ fornondissociable lactic acid and combining amendments of creeping withthese two wave-numbers (FIG. 8).

    L=(0.524×A1725+0.476+A1575)/0.00045

It has become apparent that the lactic acid concentration can becalculated according to the above equation. Glucose concentrations,lactic acid concentrations and pHs obtained from the infrared absorptionspectrum and values measured by the enzyme method and pH electrodes areshown in Table 1. Glucose concentrations and lactic acid concentrationscould be calculated from the infrared absorption spectrum measuredin-line at a high precision.

For dissociable lactic acid, absorption at 1457 cm⁻¹ 1416 cm⁻¹, 1362cm⁻¹, 1315 cm⁻¹ and 1045 cm⁻¹ other than 1575 cm⁻¹ can be utilized, andfor nondissociable lactic acid, absorption at 1237 cm⁻¹ and 1131 cm⁻¹other than 1725 cm⁻¹ can be utilized.

                  TABLE 1                                                         ______________________________________                                        Values obtained by actual measurements                                                      Lactic  Titra-                                                  Culture                                                                             Glucose acid    tion                                                    time  conc.   conc.   acidity    Absorbance/cm.sup.-1                         (h)   (g/l)   (g/l)   (ml)  pH   1035  1725  1575                             ______________________________________                                        0     19.80   0.50    1.8   6.45 0.02318                                                                             0.00000                                                                             0.00042                          4     18.47   2.07    3.1   5.92 0.02240                                                                             0.00002                                                                             0.00190                          6     15.70   4.70    5.4   4.77 0.02069                                                                             0.00055                                                                             0.00390                          8     12.93   7.32    7.6   4.25 0.01904                                                                             0.00210                                                                             0.00458                          10    10.33   9.50    9.5   4.00 0.01752                                                                             0.00379                                                                             0.00487                          15     6.17   12.60   12.1  3.73 0.01500                                                                             0.00625                                                                             0.00510                          19     3.64   14.83   14.0  3.64 0.01351                                                                             0.00790                                                                             0.00526                          27     0.40   18.06   16.8  3.53 0.01155                                                                             0.01071                                                                             0.00527                          ______________________________________                                               Values obtained by calculation from absorbance*                        Culture  Glucose Lactic      Titration                                        time     conc.   acid conc.  acidity                                          (h)      (g/l)   (g/l)       (ml)   pH                                        ______________________________________                                        0        19.80   0.45        1.7    6.48                                      4        18.50   2.05        3.1    5.84                                      6        15.65   4.80        5.4    4.71                                      8        12.90   7.31        7.6    4.20                                      10       10.37   9.57        9.5    3.97                                      15        6.17   12.66       12.2   3.77                                      19        3.68   14.74       13.9   3.68                                      27        0.42   18.00       16.7   3.55                                      ______________________________________                                    

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the infrared absorption spectrum of a glucose standardliquid from which the absorption of water is deducted.

FIG. 2 shows the relation between the absorbance from which theabsorption of water is deducted and the glucose concentration.

FIG. 3 shows the relation between the absorbance from which theabsorption of water is deducted and the lactose concentration.

FIG. 4 shows the relation between the absorbance from which theabsorption of water is deducted and the lactic acid concentration.

FIG. 5 shows the relation between the glucose concentration and theabsorbance in Example 1.

FIG. 6 shows the relation between the lactose concentration and theabsorbance in Example 2.

FIG. 7 shows the infrared absorption spectrum of a 1% lactic acidsolution with a pH of from 2 to 7 from which the absorption of water isdeducted.

FIG. 8 shows the relation between the lactic acid concentration in alogosa medium and the infrared absorbance of dissociable andnondissociable lactic acid from which that of culture time 0 isdeducted.

FIG. 9 shows the relation between the lactic acid concentration in amilk medium and the infrared absorbance of dissociable andnondissociable lactic acid from which that of culture time 0 isdeducted.

BEST EMBODIMENTS FOR CARRYING OUT THE INVENTION

Next, the present invention will be described more specificallyaccording to Examples; however, the present invention is restricted tothe following Examples by no means.

EXAMPLE 1

Lactobacillus casei was cultured stationary at 37° C. in a logosa medium(J. Inf. Dis., 110, 258-267, 1962). As typical state variables wereselected the glucose concentration, the lactic acid concentration andthe bacterial concentration. For an infrared spectrum, the continuousautomatic in-line measurement of an infrared absorption spectrum wasperformed within a range of from 1900 to 900 cm⁻¹ by a combination of aninfrared spectrometer, FT-IR FTS-65A (Bio-Rad Digilab) and an FT-IRprobe, ATR Model DPR-210 (S) (A×10 m Analytical). The accumulation andanalysis of data were investigated by means of a computer (IBM-PC). Witha view to contrasting with an infrared absorption spectrum, thequantitative determination of glucose and lactic acid was performed bythe enzyme method. As a result of it, the glucose concentration, thelactic acid concentration, the acidity and the pH value could bemeasured precisely by the method of the present invention, as describedabove.

EXAMPLE 2

Replacing the logosa medium with a milk medium, the measurement oflactic acid showing a high correlation with titration acidity, a typicalstate variable of lactic bacteria fermented milk by the infraredspectrometry was performed and investigated in the same manner as inExample 1. It has become apparent, as a result of it, that the lacticacid concentration (L) (g/l) can be calculated from the infraredabsorbance (A1725, A1575) at 1725 cm⁻¹ and 1574 cm⁻¹ according to thefollowing equation (FIG. 9):

    L=(0.524×A1725+0.476×A1575)/0.00041

Even in the milk medium with a high viscosity and adhesion, thequantitative determination of typical state variables could be performedby the method of in-line measurement wherein a probe of an infraredabsorption spectrum measuring device was fixed directly to afermentation tank. Hence, it has become apparent that the method of thepresent invention is extremely effective in the systems of fermentedmilk drinks and fermented milk, though the culture processes of themhave been monitored and controlled by titration acidity conventionally.

As described above in detail, the present invention is a method forcontrolling culture of lactic bacteria, characterized in that the methodcomprises measuring, in the step of cultivating lactic bacteria, theintensity of the infrared absorption assignable to dissociable lacticacid and that of the infrared absorption assignable to nondissociablelactic acid in the culture, the intensity of the infrared absorptionassignable to an alcoholic C-O group of glucose and/or that of theinfrared absorption assignable to an alcoholic C-O group of lactosedirectly by the FT-IR spectrometry, respectively, and calculating the pHvalue of the culture and/or the lactic acid concentration of the cultureon the basis of these measurements, and that further calculating theglucose concentration or the lactose concentration; according to thepresent invention, it becomes possible to measure various index valuesneeded for controlling the step of cultivation in a rapid, simple andprecise manner in the production of products utilizing lactic bacteriasuch as fermented milk, and thereby to monitor and control the state ofculture of lactic bacteria in the step of cultivation in a simple andrapid manner. In addition, it becomes possible by employing theATR-FT-IR spectrometry to perform the measurement of the above variousindex values in-line and to control the step of culture of lacticbacteria automatically.

What is claimed is:
 1. A method for measuring pH of culture of lacticbacteria, which comprises measuring, in the step of cultivating lacticbacteria, the intensity of the infrared absorption assignable todissociable lactic acid and that of the infrared absorption assignableto nondissociable lactic acid in the culture by the FT-IR spectrometry,and calculating the pH value of the culture according to thesemeasurements.
 2. A method for measuring pH of culture of lactic bacteriaas claimed in claim 1, characterized in that the intensity of theinfrared absorption is measured in-line by the ATR-FT-IR spectrometry.3. A method for measuring pH of culture of lactic bacteria as claimed inclaim 1, characterized in that the intensity of the infrared absorptionassignable to dissociable lactic acid is the intensity of the infraredabsorption at about 1575 cm⁻¹ and the intensity of the infraredabsorption assignable to nondissociable lactic acid is the intensity ofthe infrared absorption at about 1725 cm⁻¹.